Endolysin from bacteriophage against Geobacillus and methods of using

ABSTRACT

Bacteriophage against  Geobacillus  are provided, and methods of making and using the bacteriophage also are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) to U.S.Application No. 62/109,156, filed Jan. 29, 2015.

TECHNICAL FIELD

This disclosure generally relates to bacteriophage and endolysins fromsuch bacteriophage.

BACKGROUND

Bacteriophage destroy bacteria but are harmless to humans. They arestrain and, usually, species-specific, and they are abundant in nature,in foods, and in the intestinal tract of animals. Bacteriophage areabout 100 times smaller than bacteria, and they leave no ecologicalfootprint. Bacteriophage are generally recognized as safe (GRAS).

The lytic lifecycle of bacteriophage typically includes adsorption to abacterial cell, infection, which includes injecting their nucleic acidinto the bacterial cell, replication, maturation, and assembly ofbacteriophage inside the bacterial cell. The lytic lifecycle culminatesin lysis of the bacterial cell to release all the progeny bacteriophage.

SUMMARY

This disclosure describes bacteriophage against Geobacillus and methodsof making and using the bacteriophage.

In one aspect, an isolated bacteriophage having lytic activity againstGeobacillus is provided. Such a bacteriophage typically includes anucleic acid sequence encoding an endolysin, wherein the nucleic acidsequence has at least 95% sequence identity to a nucleic acid sequenceselected to the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, and 11. Insome embodiments, the nucleic acid sequence has at least 99% sequenceidentity to a nucleic acid sequence selected to the group consisting ofSEQ ID NOs:1, 3, 5, 7, 9, and 11. In some embodiments, the nucleic acidsequence has a sequence selected to the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, and 11. In some embodiments, the endolysin encoded bythe nucleic acid sequence has an amino acid sequence selected from thegroup consisting of SEQ ID NOs:2, 4, 6, 8, 10, and 12.

In another aspect, an isolated bacteriophage having lytic activityagainst Geobacillus is provided. Such a bacteriophage typically includesa nucleic acid sequence encoding an endolysin having at least 95%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs:2, 4, 6, 8, 10, and 12. In some embodiments,the endolysin has at least 99% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8,10, and 12. In some embodiments, the endolysin has an amino acidsequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8,10, and 12.

In still another aspect, an isolated nucleic acid molecule is provided.Typically, the nucleic acid molecule includes a nucleic acid sequencehaving at least 95% sequence identity to a nucleic acid sequenceselected to the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, and 11. Insome embodiments, the nucleic acid molecule includes a nucleic acidsequence having at least 99% sequence identity to a nucleic acidsequence selected to the group consisting of SEQ ID NOs:1, 3, 5, 7, 9,and 11. In some embodiments, the nucleic acid molecules Includes anucleic acid sequence having a nucleic acid sequence selected to thegroup consisting of SEQ ID NOs:1, 3, 5, 7, 9, and 11. In someembodiments, the nucleic acid molecule encodes a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NOs:2,4, 6, 8, 10, and 12.

In yet another aspect, a vector is provided that includes any of theisolated nucleic acids described herein. In another aspect, a host cellis provided that includes such a vector.

In one aspect, a purified polypeptide is provided. Typically, such apolypeptide includes an amino acid sequence having at least 95% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs:2, 4, 6, 8, 10, and 12. In some embodiments, the amino acidsequence has at least 99% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, and 12.In some embodiments, the amino acid sequence has an sequence selectedfrom the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, and 12.

In another aspect, a method of making a polypeptide is provided. Such amethod typically includes culturing a host cell as described hereinunder appropriate conditions.

In still another aspect, a method for reducing the number of viableGeobacillus in tobacco is provided. Such a method typically includescontacting tobacco with an effective amount of a composition comprisingany of the isolated bacteriophage described herein, any of the isolatednucleic acids described herein, any of the vectors described herein, anyof the host cells described herein, or any of the purified polypeptidesdescribed herein. In some embodiments, the tobacco is reconstitutedleaf.

In yet another aspect, reconstituted tobacco leaf is provided. Suchreconstituted tobacco leaf typically includes any of the isolatedbacteriophage described herein, any of the isolated nucleic acidsdescribed herein, any of the vectors described herein, any of the hostcells described herein, or any of the purified polypeptides describedherein.

In one aspect, a tobacco product is provided that includes suchreconstituted leaf. In some embodiments, the tobacco product is acigarette.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods and compositions of matter belong. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the methods and compositionsof matter, suitable methods and materials are described below. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the effect of the G05 endolysin on Geobacillusin culture.

DETAILED DESCRIPTION

A number of bacteria are present on tobacco growing in a field and atvarious stages of processing. Some of those bacteria are beneficial and,for example, contribute to the flavor profiles of tobacco, while some ofthose bacteria are undesirable and, for example, damage the tobacco andcontribute to unwanted tobacco-specific nitrosamines (TSNAs). Forexample, there is at least one unwanted bacteria present onreconstituted leaf (RL) which oftentimes results in a biofilm. Thepresence of a biofilm on RL can cause holes and result in significantloss of yield. The primary genus of bacteria in the biofilm (about 95%)has been identified as Geobacillus.

Bacteriophage Compositions

Isolated bacteriophages are provided herein, as well as progeny thereof.As used herein with respect to bacteriophage, “isolated” refers to abacteriophage that has been separated from the environment in which itis naturally found (e.g., that does not contain a significant amount ofother bacteriophage or of the bacterial host). As used herein, “progeny”refers to replicates of a bacteriophage, including descendants of abacteriophage created by serial passage or other methods known in theart.

In addition to bacteriophage, a bacteriophage composition also caninclude media, buffers, one or more nutrients, one or more minerals, oneor more co-factors, or any other component that is necessary to maintainviability of the bacteriophage. Additionally, components that are notrelated to the viability of the bacteriophage may be desirable in abacteriophage composition such as, without limitation, a dye or colormarker.

Bacteriophage Nucleic Acids and Polypeptides

Bacteriophage contain endolysins, a generic term for one or more enzymesthat are involved in the degradation of the peptidoglycan in thebacterial cell wall, ultimately resulting in lysis of the bacteria. Thespecificity exhibited by the bacteriophage for a particular bacteriastrain is typically attributed to the endolysin(s). Therefore, asdescribed herein, isolated bacteriophage nucleic acids are provided thatencode for the endolysins, and the purified endolysin polypeptides alsoare provided.

An endolysin gene from a bacteriophage described herein has a nucleicacid sequence shown in SEQ ID NO:1, 3, 5, 7, 9, or 11, and encodes anendolysin polypeptide having a sequence shown in SEQ ID NO:2, 4, 6, 8,10, or 12, respectively. In addition to a nucleic acid sequence shown inSEQ ID NO:1, 3, 5, 7, 9 or 11, and the polypeptide sequence shown in SEQID NO:2, 4, 6, 8, 10 or 12, nucleic acid and polypeptide sequences areprovided that differ in sequence from SEQ ID NO:1, 3, 5, 7, 9, or 11,and SEQ ID NO:2, 4, 6, 8, 10, or 12, respectively. For example, nucleicacid sequences having at least 70% sequence identity (e.g., at least75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity) to the nucleicacid sequence shown in SEQ ID NO:1, 3, 5, 7, 9, or 11 are provided.Similarly, amino acid sequences having at least 70% sequence identity(e.g., at least 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity)to the amino acid sequence shown in SEQ ID NO:2, 4, 6, 8, 10, or 12 areprovided.

To calculate the percent sequence identity of two sequences, the firstand second sequences are aligned and the number of identical matches ofnucleotides or amino acid residues between the two sequences isdetermined. The number of identical matches is divided by the length ofthe aligned region (i.e., the number of aligned nucleotides or aminoacid residues) and multiplied by 100 to arrive at a percent sequenceidentity value. It will be appreciated that the length of the alignedregion can be a portion of one or both sequences up to the full-lengthsize of the shortest sequence. It also will be appreciated that a singlesequence can align differently with other sequences and hence, can havedifferent percent sequence identity values over each aligned region. Twosequences can be aligned to determine percent sequence identity usingthe algorithm described by Altschul et al. (1997, Nucleic Acids Res.,25:3389-3402), which is incorporated into BLAST (basic local alignmentsearch tool) programs available at ncbi.nlm.nih.gov on the World WideWeb.

With respect to nucleic acids, an “isolated” nucleic acid refers to anucleic acid that is separated from other nucleic acids that are usuallyassociated with the isolated nucleic acid. Thus, an “isolated” nucleicacid includes, without limitation, a nucleic acid that is free ofsequences that naturally flank one or both ends of the nucleic acid inthe genome of the organism from which the isolated nucleic acid isderived (e.g., a cDNA or genomic DNA fragment produced by PCR orrestriction endonuclease digestion). In addition, an isolated nucleicacid molecule can include an engineered nucleic acid molecule such as arecombinant or a synthetic nucleic acid molecule. With respect topolypeptides, a “purified” polypeptide refers to a polypeptide that hasbeen separated or purified from cellular components that naturallyaccompany it. Typically, the polypeptide is considered “purified” whenit is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) bydry weight, free from the proteins and naturally occurring moleculeswith which it is naturally associated. Since a polypeptide that ischemically synthesized is, by nature, separated from the components thatnaturally accompany it, a synthetic polypeptide is “purified.”

The nucleic acids described herein (e.g., encoding the bacteriophageendolysin polypeptide) can be introduced into vectors. Vectors,including expression vectors, are commercially available or can beproduced by routine molecular biology methods. A vector containing abacteriophage nucleic acid also can have elements necessary forexpression operably linked to the bacteriophage nucleic acid, and avector further can include sequences such as those encoding a selectablemarker (e.g., an antibiotic resistance gene) and/or sequences that canbe used in purification of a polypeptide (e.g., 6×His tag).

Elements necessary for expression include nucleic acid sequences thatdirect and regulate expression of nucleic acid coding sequences such as,for example, promoter sequences. Elements necessary for expression alsocan include introns, enhancer sequences, response elements, or inducibleelements that modulate expression of a nucleic acid. As used herein,operably linked means that an element necessary for expression (e.g., apromoter and/or other regulatory element) is positioned in a vectorrelative to a nucleic acid coding sequence in such a way as to direct orregulate expression of the nucleic acid coding sequence.

Vectors containing a bacteriophage nucleic acid can be introduced intohost cells. Methods of introducing nucleic acids into host cells areknown in the art and include, without limitation, calcium phosphateprecipitation, electroporation, heat shock, lipofection, microinjection,and viral-mediated nucleic acid transfer. The term “host cell” refersnot only to the particular cell but also to the progeny or potentialprogeny of such a cell. A host cell can be any prokaryotic or eukaryoticcell. For example, nucleic acids can be expressed in bacterial cellssuch as, without limitation, E. coli, or in insect cells, yeast cells,or mammalian cells such as Chinese hamster ovary (CHO) cells or COScells. It would be appreciated by those skilled in the art that thenatural infection process of bacteriophage can be used to introduce anucleic acid or nucleic acid vector into a bacterial cell.

Methods of Using a Bacteriophage Composition and Bacteriophage NucleicAcid and Polypeptide

The bacteriophage described herein, or the bacteriophage endolysinnucleic acid or polypeptide described herein, can be used in methods ofreducing the number and/or growth of the Geobacillus bacteria onreconstituted leaf (or on any of the tobacco materials used to makereconstituted leaf), which reduces the resulting Geobacillus-producedbiofilm on the reconstituted leaf. For example, reconstituted leaf canbe contacted with the bacteriophage at any point during the process ofmaking the reconstituted leaf or after the reconstituted leaf has beenproduced. In certain instances, the tobacco material (e.g., tobaccostems, tobacco leaves, tobacco solubles) can be contacted with thebacteriophage prior to being used in or made into reconstituted leaf.Contacting reconstituted leaf (or tobacco material prior being made intoreconstituted leaf) with the bacteriophage described herein reduces theamount of biofilm present on the reconstituted leaf. Since the presenceof biofilm results in holes in the reconstituted leaf, the treatedreconstituted leaf has fewer holes, which increases yield and decreaseswaste.

Since biofilm is present in a number of different environments (e.g.,hospitals, kitchens, bathrooms, in fluid-carrying pipes (e.g., carryingwater, milk, oil, fuel, or sewage), on boat hulls, on plants or trees,in the oral cavities of animals, and/or in paper- or pulp-makingfacilities), and since at least a portion of this biofilm isGeobacillus-produced biofilm, the bacteriophage described herein can beused to reduce or eliminate the biofilm that is present in thesedifferent environments.

As used herein, a reduction (e.g., a statistically significantreduction) in the number of viable bacteria means a reduction in thenumber of bacteria that are alive and capable of, for example,replication. For example, lysed bacteria or bacteria in the process oflysing are not considered viable. The viability of bacteria can bedetermined using methods routinely used in microbiology. In addition,preventing or reducing the amount of biofilm means that the surface areacontaining biofilm is reduced or the volume of the biofilm on a surfaceis reduced relative to a “control” surface that has not been contactedwith a bacteriophage. These reductions (i.e., in the number of viablebacteria or the amount of biofilm) in the presence of any of thebacteriophage (or endolysin nucleic acid or polypeptide) describedherein are a result of the lytic activity exerted by the bacteriophage(or endolysin nucleic acid or polypeptide) on the bacteria. As usedherein, an “effective amount” of a bacteriophage or of an endolysinnucleic acid or polypeptide is an amount that results in lysis ofbacteria in an amount or at a rate that is sufficient to reduce thenumber of viable bacteria or the amount of biofilm present to a desiredlevel. As used herein, “statistically significantly” refers to a p-valueof less than 0.05 (e.g., less than 0.025 or 0.01) using an appropriatemeasure of statistical significance (e.g., a one-tailed two-samplet-test).

Methods of Obtaining Bacteriophage Compositions

Methods of obtaining bacteriophage are known in the art. See, forexample, Bacteriophages: Methods and Protocols, Volume 1: Isolation,Characterization, and Interactions (Methods in Molecular Biology), Eds,Clokie & Kropinski, 2010, Humana Press; Seeley et al., 1982, J. AppliedBacteriol., 53:1-17; Pope et al., 2011, PLoS ONE, 6:e16329; and Hendrixet al., 1999, PNAS USA, 96:2192-7. Briefly, bacteria of interest (e.g.,the target bacteria) are obtained, generally using standard culturemethods. Typically, bacteria are cultured in such as way so as toactivate the lytic phase of bacteriophage native to the bacteria andcause lysis. Following lysis of the bacteria, the bacteriophage iscollected and can be characterized using any number of known methodssuch as, without limitation, nucleic acid sequencing, electronmicroscopy, burst size, and/or attachment rate. Bacteriophage also canbe described based on their host (i.e., host profiling).

Tobacco Products

Tobacco products for adult tobacco consumers are provided that containtobacco (e.g., whole leaf, stems, cut, chopped or comminuted leaf orstem, or reconstituted leaf) that has been contacted with one or morebacteriophage (or endolysin nucleic acids or polypeptides). In someinstances, the one or more bacteriophage include the bacteriophagedescribed herein.

Under certain circumstances, the tobacco or reconstituted leaf canundergo one or more treatments in order to remove or inactivate thebacteriophage once the amount and/or growth of the respective bacteriahas reached an acceptable level. However, since bacteriophage are in thegenerally recognized as safe (GRAS) category, the bacteriophage may bepresent in the final tobacco product.

Tobacco products are known in the art and include any product made orderived from tobacco that is intended for human consumption, includingany component, part, or accessory of a tobacco product. Representativetobacco products include, without limitation, smokeless tobaccoproducts, tobacco-derived nicotine products, cigarillos, non-ventilatedrecess filter cigarettes, vented recess filter cigarettes, cigars,snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco,leaf tobacco, shredded tobacco, and cut tobacco. Representativesmokeless tobacco products include, for example, chewing tobacco, snus,pouches, films, tablets, coated dowels, rods, and the like.Representative cigarettes and other smoking articles include, forexample, smoking articles that include filter elements or rod elements,where the rod element of a smokeable material can include cured tobaccowithin a tobacco blend. In addition to the tobacco described herein(i.e., that includes one or more bacteriophages), tobacco products alsocan include other ingredients such as, without limitation, binders,plasticizers, stabilizers, and/or flavorings. See, for example, US2005/0244521, US 2006/0191548, US 2012/0024301, US 2012/0031414, and US2012/0031416 for examples of tobacco products. Suitable packaging isknown for the various types of tobacco products.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Example 1—Bacteriophage Endolysins

Geobacillus bacteriophages isolated by Micreos BV (The Netherlands),designated G01-G09, were sequenced to determine the sequence identityamong the bacteriophages and to identify the endolysin sequence fromeach. Six of the nine bacteriophages had readily identifiable endolysinsequences. Out of the 6 endolysins identified, 3 were 99% similar (G05,G08, G09). The sequences of the endolysins are shown in the attachedAppendix A.

Example 2—Cloning and Expression of the Endolysin Gene

The endolysin gene from G05 was then cloned into an expression vectorusing conventional PCR techniques, and the expression vector wastransformed into BL21-AI™ ONESHOT® Chemically Competent E. coli cells.Following expression, the protein was isolated and run on an SDS-PAGEgel.

Given the homology between the endolysin gene of G05 and the endolysingene of G08 and G09, the same PCR conditions, including primersequences, were used to clone the endolysin gene from each of the G05,G08 and G09 bacteriophages.

Example 3—Evaluating Endolysin Activity

The transformed organisms containing the endolysin gene are applied toGeobacillus in pure culture, in strong brown water (SBW), and on tobaccomatrices to test the efficacy of the endolysin on bacterial cell lysis.The purified protein is used in various tobacco matrices such asreconstituted leaf (RL) to prevent biofilm formation.

Example 4—Use of Bacteriophage in Reconstituted Leaf (RL)

Geobacillus is a biofilm-producing bacterial organism that is obligatelythermophilic and facultatively anaerobic. When it produces a biofilm ontobacco (e.g., reconstituted leaf), the congealed material interfereswith the further processing of the tobacco.

To determine the effectiveness of the bacteriophage against Geobacillusas described herein, the bacteriophage was inoculated into fresh 1/10Tryptic Soy Broth (TSB) with Yeast Extract (YE), collectively “TSBYE,”and incubated at 55° C. for up to four hours (OD between 0.5 and 0.9).The culture was then inoculated into fresh TSBYE containing either 1:1or 1:10 ratio of Phage:TSBYE, SM Buffer:TSBYE (negative control), andTSBYE. The mixtures were incubated at 55° C. overnight and then seriallydiluted 1/10 to 10⁻⁵, and plated in duplicate on Tryptic Soy Agar withYeast Extract (TSAYE). The plates were incubated at 60° C. overnight.The addition of the phage inhibited the growth of Geobacillus.

Samples from the reconstituted leaf process were collected and stored at4° C. (native) or sterilized by passing sequentially through 0.45 micronand 0.22 micron filters and stored at 4° C. (sterile). The samples werethen inoculated with mid-log phase Geobacillus, SM buffer (negativecontrol) or the bacteriophage. The samples were also supplemented withTSBYE to allow for growth. The mixtures were incubated at 55° C.overnight and then serially diluted 1/10 to 10⁻⁵, and plated induplicate on TSAYE. The plates were incubated at 60° C. overnight.Results showed that the phage inhibited the growth of Geobacillus.

To determine the effectiveness of the cloned endolysin againstGeobacillus described herein was inoculated into fresh TSBYE andincubated at 55 C for up to four hours (i.e., to an OD of between 0.5and 0.9). A 1:10 inoculation of the culture was transformed into freshTSBYE containing endolysin with a range of 0 to 200 μg/ml. The preparedsamples were then placed in a microplate reader and monitored for growthat an OD₆₀₀ for 24 hours at 50 C. Readings were taken every 20 minuteswith shaking between each reading. See FIG. 1.

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

Disclosed are methods and compositions that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed methods and compositions. These and other materials aredisclosed herein, and it is understood that combinations, subsets,interactions, groups, etc. of these methods and compositions aredisclosed. That is, while specific reference to each various individualand collective combinations and permutations of these compositions andmethods may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particularcomposition of matter or a particular method is disclosed and discussedand a number of compositions or methods are discussed, each and everycombination and permutation of the compositions and the methods arespecifically contemplated unless specifically indicated to the contrary.Likewise, any subset or combination of these is also specificallycontemplated and disclosed.

What is claimed is:
 1. A method for reducing the Geobacillus-producedbiofilm on tobacco, comprising: contacting said tobacco with acomposition comprising a polypeptide having at least 95% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO:2, 4, 6, 8, 10 and 12, wherein said tobacco is reconstitutedleaf.
 2. The method of claim 1, wherein the polypeptide has at least 99%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, 4, 6, 8, 10 and
 12. 3. The method of claim 1,wherein the polypeptide has the amino acid sequence selected from thegroup consisting of SEQ ID NO:2, 4, 6, 8, 10 and
 12. 4. The method ofclaim 1, wherein the polypeptide is encoded by a nucleic acid having atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO:1, 3, 5, 7, 9 and
 11. 5. The method of claim 1,wherein the polypeptide is encoded by a nucleic acid having at least 99%sequence identity to a sequence selected from the group consisting ofSEQ ID NO:1, 3, 5, 7, 9 and
 11. 6. The method of claim 1, wherein thepolypeptide is encoded by a nucleic acid having the sequence shown in asequence selected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9and
 11. 7. The method of claim 1, wherein the polypeptide is comprisedwithin a bacteriophage.
 8. The method of claim 1, wherein said methodreduces the surface area of said Geobacillus-produced biofilm present onsaid reconstituted leaf as compared to an untreated control.
 9. Themethod of claim 1, wherein said method reduces the volume of saidGeobacillus-produced biofilm present on said reconstituted leaf ascompared to an untreated control.
 10. The method of claim 1, whereinsaid reconstituted leaf comprises fewer holes as compared to anuntreated control.
 11. The method of claim 1, wherein said polypeptideis synthetically produced.
 12. The method of claim 1, wherein saidcontacting occurs during the process of making said reconstituted leaf.13. The method of claim 1, wherein said contacting occurs after saidreconstituted leaf has been produced.